Environmental stressors transiently activate neural systems that inhibit pain responsiveness, thereby inducing a phenomenon known as stress-induced analgesia (SIA). We have recently reported that nonopioid SIA may be mediated by the mobilization of endogenous cannabinoid lipids, such as 2-arachidonoylglycerol (2-AG), in the midbrain periaqueductal gray (PAG) [Hohmann et al., Nature 435: 1108-1112, 2005]. However, the molecular mechanisms governing 2-AG signaling in the brain under physiological conditions remain unknown. Our central hypothesis is that stress stimuli that result in nonopioid SIA activate diacylglycerol lipase (DGL) in select neurons of the PAG, causing the rapid mobilization of 2-AG. Newly- released 2-AG engages local cannabinoid receptors to induce nonopioid SIA, and is then subsequently deactivated through monoacylglycerol lipase (MGL)-mediated hydrolysis. We will test this hypothesis by developing a novel, though high-risk, methodology that unites, for the first time, in vivo virally-mediated gene transfer with targeted lipidomic analyses. Our work will identify the functional consequences of virally- mediated RNA silencing of enzymes implicated in 2-AG formation (DGL) and deactivation (MGL) using three independent complementary approaches. We will use behavioral, neuroanatomical and mass spectrometric analyses (of related lipid mediators, their precursors and metabolites) to evaluate direct and indirect consequences of DGL and MGL silencing on lipid signaling pathways and SIA. Specifically, we will determine whether virally mediated silencing of the DGL and MGL genes within the PAG (i) influences 2-AG signaling and SIA; and (ii) causes distal changes in other endocannabinoid and non-cannabinoid lipid pathways. These studies are expected to demonstrate that 1) in vivo virally-mediated RNA silencing of DGL- beta - a DGL isoform recently shown to colocalize with phospholipase C, an enzyme implicated in formation of the 2-AG precursor diacylglycerol (DAG) - suppresses 2-AG formation in the PAG and SIA, and 2) in vivo virally-mediated RNA silencing of MGL stimulates 2-AG accumulation in the PAG and SIA. Validation of our in vivo gene transfer - targeted lipidomic approach should offer a critical advantage over existing methods which rely solely on neuroanatomical measurements of mRNA or protein. The results of these studies should elucidate, for the first time, the molecular, biochemical and physiological mechanisms governing 2-AG signaling in the brain and determine the role of this endocannabinoid mediator in pain modulation. Furthermore, the results will validate targeted lipidomics as a means to quantify changes in lipid signaling that occur downstream of targeted genetic manipulations. Successful validation of our technological approach and hypotheses may facilitate development of endocannabinoid-based pharmacological and gene therapies for pain. [unreadable] [unreadable] [unreadable]